Non-mitogenic substance, its preparation and use

ABSTRACT

Proposed is a mitogen-free substance comprising cross-linked copolymers including 10 to 90 mole % of guluronic acid, the balance being made up of mannuronic acid. The substance has a molecular weight of from 10,000 to 500,000 Daltons. Also proposed are methods for preparing such substances and their use.

The invention relates to a non-mitogenic substance using guluronic acidor mannuronic acid, its preparation and use.

According to standard terminology, the term mitogenic defines thosesubstances that stimulate the division of cells which would otherwise(i.e., without the influence of this substance) not divide. Thereactions thereby triggered in the organism are abnormal and maymanifest as allergic, immunologic and pyrogenic (inflammatory)reactions. It requires no further explanation that the development andproduction of non-mitogenic substances are of the greatest interest forthe contact with living organisms in biology and medicine.

In the area of implant surgery, the application of non-mitogenicsubstances is of the greatest importance in order to suppressimmunologic reactions that would otherwise be induced causing arejection of the implant. In the treatment of numerous illnesses, livingcells are introduced into the organism which, on the one hand, aresupplied with nutrients and, on the other hand, compensate for anunderproduction or complete failure of intrinsic cells by producing cellsecretions, hormones, etc., according to the individual requirements.

One of the best known illnesses of this type is diabetes. Although thedeficiency of insulin can be adjusted by well-dosed exogenousadministration in timed intervals, it should be noted that an exact 24-hdosage in the sense of a regulating mechanism can never be achieved, sothat fluctuations and deviations from the ideal level are inevitable.This disadvantage can be corrected by implanting living cells into theorganism. In order to suppress immunologic reactions, however, the cellsmust be placed in capsules which, in turn, must meet the followingrequirements: uninhibited diffusion through the walls to permit thesupply with nutrients on the one hand, and the release of the cellsecretions, hormones, etc. on the other hand, must be ensured. Inaddition, it must be ensured that the implant does not cause immunologicreactions (i.e., it must be non-mitogenic), which would manifest in afibrotic growth on the capsule surface after a certain period of time,obstructing the required circulation and exchange of nutrients and cellsecretions.

The objective of this invention is the development, preparation andapplication of a non-mitogenic substance which does not cause allergic,immunologic and pyrogenic reactions, even after prolonged contact withthe living organism.

According to the invention, this objective is met with copolymers of amolecular weight between 10,000 and 500,000 dalton, which consist of 10to 90 mol % of guluronic acid, each supplemented to 100% with mannuronicacid.

The only component of the non-mitogenic substance may be a copolymer ofguluronic acid and mannuronic acid with a molecular composition of 10 to90 mol % of guluronic acid and the remainder being mannuronic acid.Because of the given molar ratio of both acids, the size of thecopolymer that is formed during polymerization has not been determined.Consequently, copolymers with a low molecular weight which do not meetthe requirements can also be identified. Therefore, an additionalrequirement is a molecular weight of the copolymer between 10,000 and500,000 dalton. Such molecular weights are relatively easy to obtain bydialysis using membranes and filters with suitable permeability and poreradius.

Non-mitogenicity is determined by testing the substance that waspolymerized with calcium for in vitro induction of measurableproliferation of spleen lymphocytes. For this purpose, splenocytes arecollected from mice of the inbred strain Balb-c. Cells at a count of1·10⁶ ml⁻¹ were cultivated at 37° C. in the presence of 100 μg ml⁻¹ ofthe mitogenic substance in the following growth medium: RPMI 1640medium, 10% fetal bovine serum (Boehringer, Mannheim, Germany), 2 mML-glutamine, 2 mM sodium pyruvate, non-essential amino acid (1x,Boehringer, Mannheim, Germany), 50 μM 2-mercaptoethanol, 100 units ml⁻¹of penicillin and 100 μg ml⁻¹ of streptomycin (Biochrom, Berlin,Germany). The lymphocytes showed neither a significant incorporation of¹⁴ C or ³ H thymidine into acid precipitable cellular substances(measured after 3 days) nor microscopically detectable growth(microscopic cell count after 5 to 9 days of cultivation).

In the scope of the invention, the admixture of other, in view ofmitogenicity, safe components in addition to the described copolymer isbasically optional.

Non-mitogenicity of the substance does not necessarily equalbiocompatibility in vivo. This property rather depends on other factors,such as the surface characteristics of the implant and the location ofthe implantation. Nevertheless, it is beneficial to choose compoundsthat are found to be biocompatible. Biocompatibility with regard to theinduction of foreign body reactions is tested in animal experimentsusing mice and rats. In the specific conduct of the experiment, capsulesof substances that were polymerized with barium or calcium were preparedand implanted in the peritoneum of these animals. Neither fibroticgrowth nor foreign body reactions were observed in the surroundingtissue. In the specific case, a 2% solution of the substance waspolymerized with 20 mM BaCl₂ in 0.9% NaCl, whereby gel pellets of 0.5 to1 mm diameter were formed. These were washed with 0.9% NaCl, incubatedfor two days in RPMI 1640 medium and implanted in the peritoneum of miceand rats. After three weeks, the capsules were retrieved by peritoneallavage and histologically analyzed.

An additional characteristic of the non-mitogenic substance is employedby the electrophoretic mobility and is described as follows: thesubstance consists of a component formed as a copolymer of 10 to 90 mol% of guluronic acid, with the remainder, i.e. 90% to 10%, beingmannuronic acid. The potential copolymers defined by this chemicalcomposition may be very different with regard to molecular size and therespective molecular configuration. In order to extract thenon-mitogenic substance, the different copolymers are subjected tocarrier-free electrophoresis in which the fractions of interest show amobility between 3 and 5·10⁻⁴ cm² ·V⁻¹ ·s⁻¹. Specific experiments inthis context were conducted in a 30·130·0.3 mm separation chamber, at achamber buffer temperature of 22° C., a chamber buffer composition of 15mM triethanolamine, 7.1 mM potassium acetate, 216 mM glycine (pH 7.2),11 mM glucose, 0.2 mM ethylenediamine tetraacetate, 100 units ml⁻¹ ofpenicillin, 100 μg·ml⁻¹ of streptomycin, at a conductivity of 1.9 mScm⁻¹. The buffer was pumped through the chamber at a speed of 2.5ml·h⁻¹. The pump was injected at a rate of 350 μl·h⁻¹. The fieldintensity was 100 V·cm⁻¹ (the electric current was 65 mA). As anadditional requirement, the substance must have a molecular weightbetween 10,000 and 500,000 dalton. Copolymers of these molecular weightscan be separated by diffusion through a membrane.

The inventive merit of the suggested substance is revealed in the factthat, with reference to specific experiences and experiments, expertsbelieve that mannuronic acid as a substance in itself and copolymerscontaining mannuronic acid were mitogenic (see Journal of Immunotherapy10, pp. 286-291, 1991, Raven Press, New York; TransplantationProceedings Vol. 23, No. 1 (February) 1991, pp. 758-759). The merit ofthis invention is the discovery of the inaccuracy of the above claim. Inaddition to that, alginates generally demonstrate mitogenic reactionswhich are--and this is another important discovery--to be attributed toinevitable environmental pollution due to their derivation andextraction from natural products.

Substances that are characterized by non-mitogenic properties can bederived from raw alginates by specified production procedures.

Described below are two procedures for the preparation of the previouslycited non-mitogenic substances. The first uses carrier-freeelectrophoretic processes, while in the second procedure, the substanceis obtained by chemical reaction. Common to both procedures is thestarting material of alginate which is extracted from plants, algae,bacteria, etc., and which is commercially available.

An up to 10% alginate solution is introduced into an electrophoresisbuffer with an electric conductivity of 2 mS·cm⁻¹, followed byseparation into fractions of different electrophoretic mobility, whichare collected separately. The electrophoretic mobility of interest isbetween 3 and 5·10⁻⁴ cm² ·V⁻¹ ·s⁻¹. Subsequently, separation accordingto diffusibility which differs by molecular weight, is conducted byadditional dialysis against water. The non-mitogenic substance isobtained by collection of molecular weights between 10,000 and 500,000dalton from the entire copolymer.

A chemically based production procedure is described below, in which thealginate extracted from plants, algae, bacteria, etc., was initiallycross-linked into an insoluble complex that allows washing andextraction as well as subsequent recovery. In this procedure, thealginates are precipitated by addition of Ba⁺⁺ ions or similarmultivalent ions (lead Pb, copper Cu) with a similar or higher affinityfor alginate than barium, followed by extraction with an acid, such asacetic acid, over several hours at increased temperatures. Afterwashing, further extractions are conducted in an alkaline range usingcomplex forming agents, such as citric acid. After washing in distilledwater, the gel pellets are stirred for several hours in an alcohol, suchas ethanol. The washed, but still cross-linked alginates are released byaddition of EDTA (ethylenediamine tetraacetic acid) in a highly alkalinerange. The gel pellets dissolve in this process. The result is anon-mitogenic substance derived from raw alginate.

Following is a detailed description of a chemically based productionprocess:

A 2 to 4% solution of raw alginate is filtered through a membrane filterof 0.22 μm pore size and subsequently drop-added under agitation into a5-fold volume of a 50 mM barium chloride solution, whereby gel pelletsof 1 to 5 mm in diameter are formed. After 10 to 20 minutes, the gelpellets are washed in distilled water and subsequently stirred twice for3 to 4 hours at 60 to 80° C. in 1 L each of 1M acetic acid/10 mM BaCl₂.The gel pellets are then washed on a sieve with distilled water andstirred overnight in 2×1 L of 50 mM citric acid (pH-value 8.0 with KOH),with a medium change after 4 hours. The gel pellets are re-washed indistilled water and are subsequently stirred for 2 to 3 hours in 500 mlof 80% ethanol for 1 hour each in 1 to 2 hours in distilled water.[sic.]The gel pellets are decanted through a sieve and suspended in 200ml of a 250 mM EDTA solution (pH 10.0 with KOH). After overnightstirring the pellets dissolved. The solution is dialyzed 3× againstdistilled water (pH 8.5 with KOH) for at least 4 hours and subsequentlyfreeze-dried.

Following are some examples of applications for the non-mitogenicsubstance as described according to the invention. They have in commonthat they are accepted by the animal or human body without problems andsafely prevent the induction of mitogenic reactions, even afterlong-term use.

First, the area of transplant surgery should be mentioned, in whichnon-mitogenic substances can be used for the encapsulation andimplantation of living cells without inducing immunologic reactions bythe human body. A particularly important example is the enclosure ofinsulin-generating cells (islets) in capsules of non-mitogenicsubstances which do not tend to develop fibrotic growth, even afterlong-term implantation, as was proven in animal experiments. The capsulepermits the diffusion of nutrients necessary for the maintenance of cellgrowth as well as the excretion of hormones--in the case of diabetes,this would be insulin--that are produced in this process. Thisapplication simultaneously employs the permeability for glucose, oxygen,peptide hormones--insulin is classified as such--as well as theimpermeability for larger molecules, such as in antibodies. Theimplantation of living cells (islets of Langerhans) permits an idealadjustment of the immediate hormone demand in the sense of a regulatingmechanism.

Further, non-mitogenic substances are particularly suited for use as afood additive, where they are known to aid gelling and stabilization.Statistical studies suggest connections between the use of (the current)alginates and the incidence in the development of gastrointestinal tractcancer. These currently used alginates are polluted and therefore--asestablished by the invention--mitogenic. Substitution with non-mitogenicalginates would have significant health benefits.

Finally, non-mitogenic substances can be used externally for closingwounds from injuries as well as internally for ulcers, particularlystomach ulcers. They can be safely used as dental impression compoundsand, in capsule or pellet form, serve in galenic medicine aspharmaceutical carrier substances which dissolve in the stomach orintestine, releasing the encapsulated pharmaceutical compound. In thesame manner, it can be used as a vehicle for contrast media in which,for example, air bubbles are absorbed and encapsulated, which servescontrasting in ultrasonic scans.

What is claimed is:
 1. A non-mitogenic substance comprising guluronicand mannuronic acid, in which the substance comprises copolymersconsisting of about 10 to 90 mol % of guluronic acid and about 90 to 10mol % of mannuronic acid and in which the copolymers have a molecularweight of about 10,000 to 500,000 daltons and an electrophoreticmobility between about 3 and 5·10⁻⁴ cm² ·V⁻¹ ·s⁻¹.
 2. The non-mitogenicsubstance according to claim 1, wherein the substance is biocompatible.3. A method for implanting living cells in a patient which comprisesencapsulating the living cells with the non-mitogenic substance of claim1 to form encapsulated living cells, and introducing the encapsulatedliving cells into the patient.
 4. A method for stabilizing a food whichcomprises adding the non-mitogenic substance of claim 1 to the food. 5.A method for gelling a food which comprises adding the non-mitogenicsubstance of claim 1 to the food.
 6. A method for treating an internalor external wound which comprises applying the non-mitogenic substanceof claim 1 to the wound.
 7. A method for making a dental impression of adental formation which comprises applying the non-mitogenic substance ofclaim 1 to the formation.
 8. A method for administering a pharmaceuticalto a patient which comprises combining the pharmaceutical with thenon-mitogenic substance of claim 1 to form a non-mitogenicpharmaceutical composition, and introducing the composition into thepatient.
 9. A method for the preparation of the non-mitogenic substanceof claim 1, comprising the steps of:(i) fractionating an alginatesolution of up to 10% alginate by carrier-free electrophoresis toprovide a fraction having an electrophoretic mobility between 3 and5·10⁻⁴ cm² ·V⁻¹ ·s⁻¹ ; (ii) dialyzing said fraction; and, (iii)separating alginate with a weight between 10,000 and 500,000 daltonsfrom said dialyzed fraction to recover the non-mitogenic substance. 10.A method for the preparation of the non-mitogenic substance of claim 1,comprising the steps of:(i) precipitating from an alginate solution aninsoluble alginate complex; (ii) extracting said alginate complex withan acid; (iv) extracting said alginate complex with a complex formingagent; (vi) treating said alginate complex with an alcohol; (vii)treating said alginate complex with EDTA; and, (vii) recovering thenon-mitogenic substance.
 11. The method according to claim 10, whereinthe alginate complex is precipitated by the addition of Ba⁺⁺ ions. 12.The method according to claim 10, wherein the alginate complex isprecipitated by the addition of multivalent cations.
 13. The methodaccording to claim 10, wherein the complex forming agent is citric acid.14. The method according to claim 10, wherein the steps of extractingwith a complex forming agent and the step of treating with EDTA isconducted at an alkaline pH.
 15. The method according to claim 10,wherein the alginate solution is obtained by extraction of plants, algaeor bacteria.